Method for sensitization of cancer cells for killer cell mediated lysis

ABSTRACT

The invention relates to a method of sensitizing cancer cells for a killer cell mediated lysis which involves administering to a patient an effective amount of antiestrogen and killer cells either jointly or sequentially, wherein the killer cells are selected from the group of NK cells, LAK cells and CTL cells and the antiestrogen is selected from the group of triphenylethylene class antiestrogens, such as tamoxifen or toremifene or their pharmaceutically acceptable salt.

This application is a 371 of PCT/FI94/00333 which is a continuation ofthe U.S. application Ser. No. 08/103,519, filed Aug. 9, 1993, nowabandoned.

This application is a 371 of PCT/FI94/00333 which is a continuation ofthe U.S. application Ser. No. 08/103,519, filed Aug. 9, 1993, nowabandoned.

BACKGROUND OF THE INVENTION

The invention relates to the treatment of cancer by sensitization ofcancer cells by antiestrogens for lysis with killer cells, particularlywith natural killer (NK) cells, lymphokine activated killer (LAK) cellsand cytotoxic T lymphocytes (CTL).

The treatment of human cancer with autologous lymphokine activatedkiller (LAK) cells combined with recombinant-derived lymphokine,interleukin-2, has already been attempted with encouraging results(Rosenberg, 1987) as well as the treatment with activated cytotoxic Tlymphocytes (CTL) (Fujimoto, 1992).

Nonsteroidal antiestrogens tamoxifen and toremifene belonging to thetriphenylethylene class of compounds have gained wide therapeuticapplication for the treatment of estrogen receptor positive breastcancer. Tamoxifen and toremifene inhibit estrogen-induced growth bycompetitive antagonism of tumor estrogen receptors. Antiestrogen therapyis effective in prolonging a disease-free state and overall survival ofwomen following primary surgery. Other well known triphenylethyleneclass antiestrogens are e.g. clomiphene and droloxifene(3-hydroxytamoxifen).

We have now discovered that estrogen receptor negative cancer cells aresensitized by triphenylethylene antiestrogens for lysis with NK, LAK andCTL effectors. This sensitization effect is thus not dependent on thepresence of classical estrogen receptors. The use of the combination ofantiestrogen treatment and killer cell therapy according to theinvention is expected to significantly elevate the percentage of tumorremission and cure that has already been achieved by the aboveinvestigators with killer cells alone in a minor percentage of patients.

SUMMARY OF THE INVENTION

The invention relates to a method of sensitizing cancer cells for akiller cell mediated lysis which involves administering to a patient aneffective amount of antiestrogen and killer cells either jointly orsequentially, wherein the killer cells are selected from the group of NKcells, LAK cells and CTL cells and the antiestrogen is selected from thegroup of triphenylethylene class antiestrogens, such as tamoxifen ortoremifene or their pharmaceutically acceptable salt. Alternatively,killer cells may be induced in a host by one of known immunostimulationmethods during the first treatment period and thereafter administeringan effective amount of antiestrogen during the second treatment period.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph of a Winn assay with untreated, tamoxifen (TX) treatedand toremifene (TO) treated P815 tumor cells.

FIGS. 2a, 2b and 2c are the survival curves for the experiment of FIG.1.

FIG. 3 is a graph of the effect of oral TX and TO treatment alone orwith LAK treatment on P815 tumor growth.

FIGS. 4a, 4b and 4c are the survival curves for the experiment of FIG.3.

FIG. 5 is a graph of a winn assay with untreated, tamoxifen (TX) treatedand toremifene (TO) treated P815 tumor cells.

FIGS. 6a, 6b and 6c are the survival curves for the experiment of FIG.5.

FIG. 7 is a graph demonstrating the immunotherapy results of P815mastocytoma with TX or TO and LAK cells.

FIGS. 8a, 8b and 8c are the survival curves for the experiment of FIG.7.

FIG. 9 is a graph demonstrating the immunotherapy results of P815mastocytoma with TX or TO given by gavage and LAK cells.

FIGS. 10a, 10b and 10c are the survival curves for the experiment ofFIG. 9.

DETAILED DESCRIPTION OF THE INVENTION

1. Detection of killer cells

The prerequisite for success of the treatment according to the inventionis the presence of killer cells (e. g. NK, LAK or CTL) capable ofdestroying the patient's cancer cells in the body when the adjuvanttherapy with an antiestrogen, e. g. tamoxifen (TX) or toremifene (TO),is initiated. Ideally, a suspension of cells would be prepared from thepatient's tumor by digestion with collagenase as described by Freshney(1976) and used as target cells. Some of the targets will then bepretreated for 4 hours with either TX or TO while others are leftuntreated, and both preparations are labeled with ⁵¹ Cr. Patient'speripheral blood mononuclear cells depleted of adherent cells are usedas effectors in ⁵¹ Cr release assay. Surplus tumor cells may be storedin tissue culture medium, such as RPMI-1640, supplemented with 10% serum(preferably autologous) and 10% dimethylsulphoxide in liquid nitrogenfor later use. If this approach is not feasible, one may be able to usecell lines as targets, such as K652, which is suitable for the detectionof NK activity, or lines derived from the same type of cancer thepatient is suffering from. The presence of tumor infiltratinglymphocytes (TIL) in biopsy specimen may be taken as indication for thepresence of host derived killer cells. Additional procedures that areused to detect cell mediated immunity and could be of use are: delayedtype hypersensitivity skin reactions elicited by tumor antigen/ extract;inhibition of peripheral leukocyte migration by tumor antigen/extract;and the proliferative response of CD8+ T lymphocytes to tumorantigen/extract in vitro.

Once it has been established by any one of the above procedures that thepatient harbours killer cells capable of killing his/her cancer,antiestrogen therapy will be initiated until tumor regression isachieved. If killer cells are not present they should be induced in ahost by reduction of tumor burden or by immunostimulation methods asdescribed below.

2. Reduction of tumor burden

Most patients with overwhelming tumor burden will not have detectablekiller cells in their circulation. Here the first task would be toreduce tumor burden, if possible, by surgery, radiation or chemotherapy.There are numerous animal experiments and observations on patients toindicate that the reduction of tumor burden frequently leads to theappearance of killer cells, both in the circulation and in the tumoritself. Once the killer cells are detectable, the adjuvant therapy withthe antiestrogen would commence.

3. Immunostimulation

If the reduction of tumor burden does not lead to the spontaneousappearance of killer cells in the patient, the stimulation of suchkiller cells may be attempted. Some of the approaches that can be usedare listed below:

(a) Vaccination.

(b) Treatment with cyclophosphamide, which is known to inhibitsuppressor immune mechanisms, and thus to enhance cellular immunity.Similarly, indomethacin interferes with the suppressive effect ofmacrophages.

(c) By the application of cytokines, such as interleukin-2.

(d) By the use of adjuvants stimulating cell mediated immunity such asmuramyl peptide and synthetic bacterial wall analogues (Utsugi et al,1991).

(e) By dietary factors, such as vitamin A (Gergely et al, 1988).

Suitable vaccination procedures are described by Livingston, 1992; Changet al, 1993 and Hoover et al 1993. Cyclophosphamide treatment isdescribed, for example, by Komatsumoto et al, 1991 and Kassabov andStoychkov, 1991. Indomethacin treatment is described, for example, bySaarloos et al, 1993 and Aso et at, 1992.

The usual therapeutic dose for cyclophosphamide is 50 mg daily and forindomethacin 50 mg daily. Cytokines, such as interleukin-2 (IL-2), mayalso be used for immunostimulation as described e. g. in Yoshino et al.(1991). Yoshino et al. treated patients with low dose IL-2 (2×10⁶ Japanreference units s. c. initially, which dose was decreased to half oneach consecutive day for 6 days) which resulted in significantactivation of NK and LAK cells.

Once killer cells have been induced, target sensitization withantiestrogen therapy would commence.

4. Joint Therapy with Killer Cells

Administering in vitro activated killer cells of NK, LAK or CTL type andantiestrogen either jointly or sequentially is within the scope of theinvention. Antiestrogen is selected from the group of triphenylethyleneantiestrogens. The preferred antiestrogen is toremifene or tamoxifen ortheir pharmaceutically acceptable salt. Our experiments show that suchcombination therapy has a definite advantage in comparison with eitherdrug treatment or killer cell therapy alone. According to ourexperiments target cell sensitization is optimally exploited at thestage when killer cells are already generated by the patient, oralternatively, when the killer cells are applied jointly with the drugs.

For the generation of LAK cells, patient's mononuclear lymphocytes arecultured with interleukin-2. The mononuclear lymphocytes are collectedas described in Rosenberg et al (1987). The procedure can be used inorder to collect about 1×10⁸ to 1×10¹¹ mononuclear cells per patient.The lymphocytes can then be separated from the adherent cells usingknown procedures. The resulting mononuclear lymphocytes are cultured inany appropriate media such as RPMI-1640 supplemented with humanrecombinant interleukin-2, for example at 37° C. for three to five days.The resulting LAK cells are isolated and suspended in media suitable forintravenous infusion into the patient. The LAK cells are administeredpreferably once every day or every other day for from one to five dailydoses. Preferably from 1×10⁸ to 1×10¹⁰ LAK cells are administered dailyon each of three days over a period of from 3 to 10 days. The patientmay require from one to four such regimens sequentially. NK and CTLcells may be administered in a similar fashion. Further guidelines forNK and CTL therapy may be obtained from the publications of Rosenberg etal. (1987) and Fujimoto (1992).

The doses for TX and TO and treatment schedule that are currently in usewould be applied in therapy according to the invention. For TX ansuitable oral dose is 20-40 mg given daily (Anderson et al. , 1981). Thesuitable oral dose for TO is 40-60 mg, but can be as high as 240 mgdaily (Hietanen et al. , 1990). Preferably the antiestrogen treatment isstarted as soon as the killer cells are generated or infused into thepatient. The antiestrogen therapy and killer cell therapy are thenmaintained jointly, optimally until the complete cure of the patient. Itis also within the scope of the invention to apply the antiestrogentherapy and killer cell therapy sequentially, i. e. killer cell therapyis commenced immediately after discontinuation of antiestrogen therapy.However, the joint therapy is the preferred procedure.

In conclusion, the treatment procedure of using TX or TO to enhance cellmediated host defense according to the invention is preferably asfollows:

(a) Tests are performed for the detection of killer cells in thepatient;

(b) if killer cells are not present they are induced by known methods,or

(c) killer cells are generated in vitro and infused to the patient;

(d) TX or TO adjuvant therapy is applied as soon as the killer cells aregenerated or infused into the patient, preferably after thedetermination of the sensitizing effect of these drugs on the targetcell in question for lysis by the effector cells available;

(e) during therapy with the sensitizing drugs the patient is followedclosely for effectiveness.

Various modifications can be made in the therapeutic method of thepresent invention without departing from the spirit and scope thereof.The embodiments described herein are for the purpose of illustrating theinvention but are not intended to limit it.

Example 1.

The effect of toremifene (TO) and tamoxifen (TX) on target cell lysis bynatural killer (NK) cells, lymphokine activated killer (LAK) cells, andcytotoxic T lymphocytes (CTL) was studied. Rat spleen cells were used aseffector cells either without activation (NK cells), or after treatmentwith human IL-2 (LAK), or after stimulation with the Nb2 rat lymphoma inmixed cultures (CTL). The Yac-1 murine lymphoma was used as a target forNK cells, the P815 mastocytoma for LAK cells and Nb2 cells for CTL.Target and effector cells were pretreated with TO or TX, atconcentrations from 1 nM to 5 μM, for 4 hours and then washed. Targetcells were labeled with ⁵¹ Cr for 1 hr, then washed. The specific ⁵¹ Crrelease was determined after 4 hours of incubation at 37° C., 5% CO₂(target/effector 1:25). The results presented in Tables 1-3 show that TOand TX enhanced target cell lysis in a dose dependent fashion with NK,LAK or CTL effectors, if the target cells were treated for 4 h prior tothe cytotoxic reaction. The pretreatment of effector cells had no suchenhancing effect, but TO or TX treated effectors lysed treated targetsas efficiently as did nontreated effectors.

                  TABLE 1                                                         ______________________________________                                        The effect of tamoxifen (TX) and toremifene (TO)                              on NK cell mediated cytotoxicity.                                                      Percent specific .sup.51 Cr release                                  n    Treatment Target treated                                                                            Effector treated                                                                       Both treated                              ______________________________________                                        7    none      10 ± 1   10 ± 1                                                                              10 ± 1                                 4    TX 1 μM                                                                              74 ± 2     1 ± 0.2                                                                           77 ± 2                                 4    TX 100 nM 68 ± 1   0.3 ± 0                                                                             74 ± 2                                 2    TX 10 nM  61 ± 1   0 ± 0 62 ± 1                                 2    TX 1 nM   30 ± 2   0 ± 0 42 ± 2                                 4    TO 5 μM                                                                              65 ± 3   1.3 ± 0.7                                                                           68 ± 3                                 4    TO 1 μM                                                                              54 ± 3   1.0 ± 0.4                                                                           59 ± 3                                 4    TO 100 nM 48 ± 2   0.3 ± 0                                                                             52 ± 1                                 3    TO 10 nM  40 ± 1   0 ± 0 41 ± 2                                 2    TO 1 nM   25 ± 3   0 ± 0 30 ± 2                                 ______________________________________                                         Target cells: Yac1 murine lymphoma                                            Effector cells: Spleen lymphocytes of female Fishner rats                

                  TABLE 2                                                         ______________________________________                                        The effect of tamoxifen (TX) and toremifene (TO)                              on target cell destruction by cytolytic                                       lymphocytes generated in mixed cultures                                                Percent specific .sup.51 Cr release                                  n    Treatment Target treated                                                                            Effector treated                                                                       Bath treated                              ______________________________________                                        2    none      11 ± 1   11 ± 1                                                                              11 ± 1                                 2    TX 1 μM                                                                              76 ± 2   8 ± 0 82 ± 2                                 2    TX 100 nM 45 ± 2   5 ± 1 52.5 ± 1.5                             2    TO 5 μM                                                                              62 ± 2   5.5 ± 0.5                                                                           61 ± 1                                 2    TO 1 μM                                                                              52 ± 2   3.5 ± 0.5                                                                           54 ± 2                                 2    TO 100 nM 31 ± 1   1.5 ± 0.5                                                                           35 ± 3                                 2    TO 10 nM  25 ± 1   0 ± 0 22 ± 2                                 2    TO 1 nM     16 ± 1.5                                                                             0 ± 0 14.5 ± 3.5                             ______________________________________                                         Target cells: Nb2 rat lymphoma                                                Effector cells: Cytotoxic Tlymphocytes (CTL) generated in mixed cultures,     harvested on day 5                                                            Responder: Spleen cells of Fischer rats. Stimulator: Nb2 cells (thymic        lymphoma of Noble rats).                                                 

                  TABLE 3                                                         ______________________________________                                        The effect of tamoxifen (TX) and toremifene (TO)                              on LAK cell mediated cytotoxicity.                                                     Percent specific .sup.51 Cr release                                  n    Treatment Target treated                                                                            Effector treated                                                                       Both treated                              ______________________________________                                        3    none      10 ± 2   10 ± 2                                                                              10 ± 2                                 2    TX 1 μM                                                                              78 ± 0   2 ± 0 84 ± 1                                 1    TX 100 nM 72 ± 0   2 ± 0 82 ± 0                                 3    TO 5 μM                                                                              75 ± 4     2 ± 0.3                                                                           77 ± 2                                 3    TO 1 μM                                                                              66 ± 3   0 ± 0 70 ± 0                                 3    TO 100 nM 59 ± 1   0 ± 0 61 ± 1                                 3    TO 10 nM  48 ± 1   0 ± 0   50 ± 0.3                             3    TO 1 nM   36 ± 2   0 ± 0 43 ± 2                                 ______________________________________                                         Target cells: P815 (NKresistant murine mastocytoma)                           Effectar cells: Spleen cells of Fischer rats were cultured in medium          supplemented with 500 U/ml riL2 for 6 days at 37° C. and 5%            CO.sub.2.                                                                

Example 2.

For the generation of LAK cells, 2×10⁶ /ml of spleen cells from femaleDBA/N2 mice were cultured for 5 days in RPMI-1640 medium supplementedwith 10%FCS, 5×10⁻⁵ M 2-mercaptoethanol and 500 U/mI of humanrecombinant interleukin-2 at 370C in humidified air containing 5% CO₂.The LAK cells generated killed, at 1:25 ratios, 10 % of P815 cells, 52 %of tamoxifen (TX) treated (1 μM, 4 h) and 51 % of toremifene (TO)treated (5 μM, 4 h) cells in the ⁵¹ Cr assay. The same effector cellswere then mixed with the same untreated or drug treated P815 cells at1:25 target:effector cell ratios and injected s. c. to syngenic DBA/2recipients (Winn assay). Groups of 4 female animals were used and tumorgrowth was followed until all controls succumbed to neoplasia. Furtherdetails of the experimental design and tumor size are presented inFIG. 1. In this figure only those animals that developed a tumor areincluded. The survival curves are presented in FIGS. 2 a, b and c.

As is obvious from the results, drug treatment alone or LAK cells aloneexerted some tumor inhibition, and when both drug treatment and effectorcells were applied, tumor inhibition was enhanced. For instance, tumorcells exposed to TX or LAK cells killed all the animals, whereas oneanimal was protected in the TX+LAK group. Pretreatment with TO protected1 of 4 animals, whereas TO+LAK protected 2 of 4 animals. Also, it isclear from FIG. 1 that there is a marked retardation of tumor growth inthe drug+LAK treated groups between days 17-23.

Example 3.

In the previous experiment there was only a brief exposure of the tumorcells to the drug (4 h) and to LAK cells, which explains that tumorretardation was limited to the early period in those animals whicheventually grew the tumor. In the next experiment the same basic designwas used , but this time drug treatment was given in the drinking water(TX, 100 μg/ml and TO, 400 μg/ml) starting 2 days prior to tumorinjection and maintained throughout the experiment. Again, some of thegroups received tumor cells mixed with LAK cells at 1:25 ratio andtreated further with the same dose of LAK cells injected to the tumorsite weekly for 3 weeks. This time significant tumor inhibition occurredin the animals treated with either drug alone and also in thosereceiving LAK cells only, as can be seen FIG. 3. Again, tumor growth wasthe slowest in those groups with both drug and LAK treatment. Moreover,there is one animal in the TX+LAK group and also in the TO and TO+LAKgroup which is tumor free at this time, as can be seen in FIG. 4 a, band c. Therefore, with continuous treatment we succeeded in slowingtumor growth considerably although the proportion of tumor free animalsdid not increase.

Example 4.

The experiment of Example 2 was repeated with increased amount of LAKcells (1:50 ratio) mixed with drug treated or nontreated tumor cells andapplied s. c. to animals. The results are shown in FIG. 5. The survivalcurves for FIG. 5 are shown in FIG. 6a, b and c. Again, as in previousexperiments, there are some tumor free animals in the treated groups,especially when drug treatment is combined with killer cells.

Example 5.

In this immunotherapy experiment all groups received the same dose oftumor cells s. c. and 12 days later when the tumor was clearly palpablein all animals, drug treatment in the drinking water was initiated.Treatment with LAK cells was started 2 days later and given 3 times, 4days apart, as indicated in FIG. 7. Drug treatment was maintainedthroughout the experiment. As is obvious from FIG. 7, tumor growth wasmarkedly retarded by drug treatment combined with killer cells. This isalso reflected in the survival curves, which are presented in FIG. 8a, band c. All the controls succumbed to neoplasia by day 26, whereas allthe animals are still alive on day 28 in the TO+LAK group. There is onetotal regression in TX+LAK and one partial regression in TO+LAK treatedgroups (the tumor volume decreased to 10 times less than the originalvolume).

Example 6.

DBA/2 female mice were injected s. c. with 10⁴ P815 tumor cells. Whenthe tumors were approximately 0.5 cm diameter, drug treatment wasintroduced (either TX or TO, 10 mg/kg/day by gavage). Two days later25×10⁴ LAK cells were given i.p., LAK was injected three times more,four days apart. Drug treatment was maintained from day 14 until day 30.The results are shown in FIG. 9. Again, tumor growth was markedlyretarded by drug treatment combined with killer cells. The survivalcurves are presented in FIG. 10 a, b and c. There was one totalregression in TX+LAK and one partial regression in TO+LAK treated group(the tumor volume decreased to 10 times less than the original volume).Furthermore there was one partial regression both in TX group and TOgroup.

References Anderson M. et al., J. Natl. Cancer Inst. , 83, 1013-1017,1991.

Freshney R., Monolayer cultures, In: Human tumours in short termculture. Techniques and clinical applications, PP Dendy, ed. AcademicPress, London, 1976.

Fujimoto S. et al., Hum. Cell., 5, 247-55, 1992.

Hietanen T. et al., Breast Res. Treat., 16(Supp), S37-S40, 1990.

Rosenberg S. et al., N. Eng. J. Med., 316, 889-97, 1987.

Yoshino I. et al., Cancer Res., 51, 1494-8, 1991.

Hoover H. et al., J. Clin. Oncol., 11, 3, 390-9, 1993.

Chang A. et al., Cancer Res., 53, 5, 1043-50, 1993.

Livingston P., Curr. Opin. immunol., 4, 5, 624-9, 1992.

Komatsumoto M. et al., Cancer Immunol. Immunother., 33, 279-84, 1991.

Stoychkov J. and Kassabov K., Cancer Immunol. Immunother., 33, 307-313,1991.

Saarloos M. et al., Cancer Res., 52, 23, 6452-62, 1992.

Aso H. et al., Microbiol Immunol., 36, 10, 1087-97, 1992.

Utsugi T. et al., Cancer Immunol. Immunother., 33, 285-292, 1991.

Gergely P. et al., Acta Med. Hung., 45, 3-4, 307-11, 1988.

We claim:
 1. A method for inducing or enhancing killer cell mediatedlysis of cancer cells which do not express estrogen receptors (ER-) in acancer patient comprising administering to said cancer patient (i) aneffective amount of a triphenylethylene antiestrogen, and (ii) aneffective amount of killer cells selected from the group consisting ofNK cells, LAK cells and CTL cells, wherein the administration of (i)said triphenylethylene antiestrogen and (ii) said killer cells iseffected jointly or sequentially, and wherein the amount of saidtriphenylethylene antiestrogen and said killer cells is effective toinduce or enhance killer cell mediated lysis of cancer cells which donot express estrogen receptors which are contained in said patient, andfurther wherein the amount of said triphenylethylene antiestrogenrelative to said killer cells is sufficient to sensitize said cancercells which are contained in said patient such that greater numbers ofsaid cancer cells are lysed by killer cell mediated lysis in comparisonto when either said killer cells or antiestrogen are administeredsingularly.
 2. The method of claim 1, wherein said triphenylethyleneantiestrogen is selected from the group consisting of tamoxifen,toremifene and pharmaceutically acceptable salts thereof.
 3. The methodof claim 2, wherein said triphenylethylene antiestrogen is tamoxifen ora pharmaceutically acceptable salt thereof.
 4. The method of claim 2,wherein said triphenylethylene antiestrogen is toremifene or apharmaceutically acceptable salt thereof.
 5. The method of claim 3,wherein said tamoxifen or pharmaceutically acceptable salt is orallyadministered at a daily amount ranging from about 20 to 40 mg.
 6. Themethod of claim 4, wherein said toremifene or pharmaceuticallyacceptable salt is orally administered in a daily amount ranging fromabout 40 to 240 mg.
 7. The method of claim 1, wherein said methodresults in tumor inhibition.
 8. The method of claim 7, wherein saidmethod results in greater tumor inhibition that when the same amount oftriphenylethylene antiestrogen or killer cells is administeredsingularly.
 9. The method of claim 1, wherein said LAK cells areadministered daily at a dosage of about 10×10⁸ to 1×10¹⁰ cells.
 10. Amethod for inducing or enhancing killer cell mediated lysis of cancercells which do not express estrogen receptors in a cancer patientcomprising the following steps:(i) stimulating the immune system of acancer patient to induce or enhance the number of killer cells producedby said cancer patient by admininistering an agent that stimulates theimmune system; and (ii) administering to said cancer patient an amountof a triphenylethylene antiestrogen which is sufficient to induce orenhance killer cell mediated lysis of cancer cells which do not expressestrogen receptors which are contained in said patient and furtherwherein the amount of said triphenylethylene antiestrogen is sufficientto sensitize said cancer cells which are contained in said patient suchthat greater numbers of said cancer cells are lysed by killercell-mediated lysis in comparison to when said triphenylethyleneantiestrogen or said killer cell stimulation is administered or effectedsingularly.
 11. The method of claim 10, wherein said triphenylethyleneantiestrogen is selected from the group consisting of tamoxifen,toremifene and pharmaceutically acceptable salts thereof.
 12. The methodof claim 10, wherein the immune system of said cancer patient isstimulated to produce killer cells by a method selected from the groupconsisting of treatment with cyclophosphamide, administration ofantigen, administration of cytokine, administration of adjuvant, andadministration of vitamin A.
 13. The method of claim 10, wherein theimmune system of said cancer patient is stimulated to produce killercells by the administration of IL-2.
 14. The method of claim 11, whereinthe immune system of said cancer patient is stimulated to produce killercells by the administration of IL-2.
 15. A method for enhancing killercell mediated lysis of tumor cells which do not express estrogenreceptor (ER-) in a cancer patient comprising:(i) detecting the presenceor absence of killer cells in a cancer patient which are capable oflysing tumor cells which do not express estrogen receptors in saidpatient; and (ii) if said killer cells are present, administering tosaid cancer patient an amount of at least one triphenylethyleneantiestrogen which is sufficient to enhance killer cell mediated lysisof said tumor cells in said patient.
 16. The method of claim 1, whereinthe killer cells are natural killer (NK) cells.
 17. The method of claim11, wherein the killer cells are lymphokine activated killer (LAK)cells.
 18. The method of claim 1, wherein the killer cells are cytotoxicT lymphocyte (CTL) cells.
 19. The method of claim 10, wherein the killercells are natural killer (NK) cells.
 20. The method of claim 10, whereinthe killer cells are cytotoxic T lymphocyte (CTL) cells.
 21. The methodof claim 10, wherein the killer cells are lymphokine activated killer(LAK) cells.
 22. The method of claim 1, which further includes an invitor assay prior to treatment to determine weather the patient harborskiller cells capable of destroying his/her cancer cells.
 23. The methodof claims 22, wherein said in vitro assay comprises obtaining asuspension of tumor cells obtained from the patient and contacting thetumor cells with killer cells obtained from the patient in acytotoxicity assay.
 24. The method of claim 22, which further includesan in vitro assay prior to treatment to determine whether the tumorcells obtained from the patient can be lysed after treatment withtriphenylethylene antiestrogens by autologous killer cells.
 25. Themethod of claim 24, wherein said further in vitro assay comprisesobtaining a suspension of tumor cells form the patient, pretreating thetumor cells with a triphenylethylene antiestrogen and contacting thepretreated tumor cells with killer cells obtained from the patient in acytotoxicity assay.